Detection of amplified or deleted chromosomal regions

ABSTRACT

The present invention relates to in situ hybridization methods for the identification of new chromosomal abnormalities associated with various diseases. In particular, it provides probes which are specific to a region of amplification in chromosome 20.

This invention was made with Government support under Contract No.W-7405-ENG-48 awarded by the Department of Energy and Grant No. CA-45919awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Chromosome abnormalities are often associated with genetic disorders,degenerative diseases, and cancer. In particular, the deletion ormultiplication of copies of whole chromosomes or chromosomal segments,and higher level amplifications of specific regions of the genome arecommon occurrences in cancer. See, for example Smith, et al., BreastCancer Res. Treat., 18: Suppl. 1:5-14 (1991, van de Vijer & Nusse,Biochim. Biophys. Acta. 1072:33-50 (1991), Sato, et al., Cancer. Res.,50: 7184-7189 (1990). In fact, the amplification and deletion of DNAsequences containing protooncogenes and tumor-suppressor genes,respectively, are frequently characteristic of tumorigenesis.Dutrillaux, et al., Cancer Genet. Cytogenet., 49:203-217 (1990). Clearlythe identification of amplified and deleted regions and the cloning ofthe genes involved is crucial both to the study of tumorigenesis and tothe development of cancer diagnostics.

The detection of amplified or deleted chromosomal regions hastraditionally been done by cytogenetics. Because of the complex packingof DNA into the chromosomes, resolution of cytogenetic techniques hasbeen limited to regions larger than about 10 Mb; approximately the widthof a band in Giemsa-stained chromosomes. In complex karyotypes withmultiple translocations and other genetic changes, traditionalcytogenetic analysis is of little utility because karyotype informationis lacking or cannot be interpreted. Teyssier, J. R., Cancer Genet.Cytogenet., 37:103 (1989). Furthermore conventional cytogenetic bandinganalysis is time consuming, labor intensive, and frequently difficult orimpossible.

More recently, cloned probes have been used to assess the amount of agiven DNA sequence in a chromosome by Southern blotting. This method iseffective even if the genome is heavily rearranged so as to eliminateuseful karyotype information. However, Southern blotting only gives arough estimate of the copy number of a DNA sequence, and does not giveany information about the localization of that sequence within thechromosome.

Comparative genomic hybridization (CGH) is a more recent approach toidentify the presence and localization of amplified/deleted sequences.See Kallioniemi, et al., Science, 258:818 (1992). CGH, like Southernblotting, reveals amplifications and deletions irrespective of genomerearrangement. Additionally, CGH provides a more quantitative estimateof copy number than Souther blotting , and moreover also providesinformation of the localization of the amplified or deleted sequence inthe normal chromosome.

Generally, where detection of deletions or amplifications is limited tothe loss or gain of one copy of a sequence, the resolution of prior artmethods may be limited. New techniques which provide increasedsensitivity, more precise localization of the affected DNA sequence, andmore quantitative estimate of copy number, even in samples of mixednormal and tumor cells is particularly desirable. The present inventionprovides these and other benefits.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for detectingchromosome abnormalities (such as deletions and amplifications) in apreselected chromosome. The methods comprise providing a mapped libraryof labeled probes specific to the chromosome; contacting a chromosomesample from a patient with the library under conditions in which theprobes bind selectively with target polynucleotide sequences in thesample to form hybridization complexes; detecting the hybridizationcomplexes; and determining the copy number of each complex.

If the selected chromosome is human chromosome 20 or 17, the preferredlibraries are as shown in Table 1 and Table 2, respectively. The methodsare typically carried out using fluorescent in situ hybridization andthe probes are labeled with digoxigenin or biotin. The probes can beused to detect the target sequences in interphase nuclei in the sample.A reference probe which binds selectively to a sequence within thecentromere of the preselected chromosome can be used as a control.

Also provided are methods of detecting specific abnormalities disclosedhere. In particular, methods of detecting an amplification at aboutposition FLpter 0.85 on human chromosome 20 are disclosed. The methodscomprise contacting a chromosome sample from a patient with acomposition consisting essentially of one or more labeled nucleic acidprobes each of which binds selectively to a target polynucleotidesequence at about position FLpter 0.85 on human chromosome 20 underconditions in which the probe forms a stable hybridization complex withthe target sequence; and detecting the hybridization complex. The probesused preferably comprise polynucleotide sequences from cS20.10A1, pkcS20.10B5, cS20.10H1, or cS20.10E2.

Also provided are compositions comprising nucleic acid probes which bindselectively to a target polynucleotide sequence at about FLpter 0.85 onhuman chromosome 20. The probes may be labeled for use in the methods ofthe invention.

The invention further provides kits for the detection of anamplification at about position FLpter 0.85 on human chromosome 20. Thekits comprise a compartment which contains a nucleic acid probe whichbinds selectively to a target polynucleotide sequence at about FLpter0.85 on human chromosome 20. The probes preferably comprisepolynucleotide sequences from cS20.10A1, cS20.10B5, cS20.10H1, andcS20.10E2. The may further comprise Texas red avidin and biotin-labeledgoat anti-avidin antibodies.

DEFINITIONS

A "chromosome sample" as used herein refers to a tissue or cell sampleprepared for standard in situ hybridization methods described below. Thesample is prepared such that individual chromosomes remain substantiallyintact and typically comprises metaphase spreads or interphase nucleiprepared according to standard techniques.

As used herein a "probe" is defined as a polynucleotide (either RNA orDNA) capable of binding to a complementary target cellular geneticsequence through one or more types of chemical bonds, usually throughhydrogen bond formation. It will be understood by one of skill in theart that probes will typically substantially bind target sequenceslacking complete complementarity with the probe sequence depending uponthe stringency of the hybridization conditions. The probes arepreferably directly labelled as with isotopes or indirectly labelledsuch as with biotin to which a streptavidin complex may later bind. Byassaying for the presence or absence of the probe, one can detect thepresence or absence of the target. Probes of the invention willtypically be between about 20 kb to about 60 kb, usually between about30 and 50 kb.

A "composition consisting essentially of one or more probes each ofwhich binds selectively to a target polynucleotide sequence" refers to acollection of one or more probes which bind substantially to the targetsequence and nowhere else in the target chromosome or genome and whichallow the detection of the presence or absence of the target sequence.Such a composition may contain other nucleic acids which do notmaterially affect the detection of the target sequence. Such additionalnucleic acids include reference probes specific to a sequence in thecentromere in the chromosome.

"Bind(s) substantially" refers to complementary hybridization between anoligonucleotide and a target sequence and embraces minor mismatches thatcan be accommodated by reducing the stringency of the hybridizationmedia to achieve the desired detection of the target polynucleotidesequence.

"Hybridizing" refers the binding of two single stranded nucleic acidsvia complementary base pairing.

"Nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymerin either single- or double-stranded form, and unless otherwise limited,would encompass known analogs of natural nucleotides that can functionin a similar manner as naturally occurring nucleotides.

One of skill will recognize that the precise sequence of the particularprobes described herein can be modified to a certain degree to produceprobes that are "substantially identical" to the disclosed probes, butretain the ability to bind substantially to the target sequences. Suchmodifications are specifically covered by reference to the individualprobes herein. The term "substantial identity" of polynucleotidesequences means that a polynucleotide comprises a sequence that has atleast 90% sequence identity, more preferably at least 95%, compared to areference sequence using the methods described below using standardparameters.

Two nucleic acid sequences are said to be "identical" if the sequence ofnucleotides in the two sequences is the same when aligned for maximumcorrespondence as described below. The term "complementary to" is usedherein to mean that the complementary sequence is identical to all or aportion of a reference polynucleotide sequence.

Sequence comparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two sequences over a "comparisonwindow" to identify and compare local regions of sequence similarity. A"comparison window", as used herein, refers to a segment of at leastabout 20 contiguous positions, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted by thelocal homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman and Wunsch J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearsonand Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), bycomputerized implementations of these algorithms. These references areincorporated herein by reference.

"Percentage of sequence identity" is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to the same sequence under stringentconditions. Stringent conditions are sequence dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength and pH. The Tm isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration is atleast about 0.02 molar at pH 7 and the temperature is at least about 60°C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the physical location of chromosome 20 specific cosmids.The locations, determined by metaphase Fish and digital image analysis,are shown as the mean of the Flpter (±sem).

FIG. 2 shows the spot numbers of the different mapped, chromosome20-specific cosmids (see FIG. 1) counted in interphase BT474 breastcancer cells. A region around FLpter-0.85 is heavily amplified. Thisregion is therefore likely to contain a (proto)-oncogene.

FIG. 3 shows the physical locations on chromosome 17 of 40 cosmidsselected from the library LA17NC01. The locations, determined bymetaphase FISH and digital image analysis, are shown as the mean of theFLpter (±sem).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods and probe libraries useful fordetecting chromosome abnormalities in situ. In particular the inventionprovides a means of identifying the presence of multiplications ordeletions in chromosomes and rapidly identifying the chromosomal regionsinvolved in those deletions or multiplications.

Detection and Localization of Chromosomal Abnormalities

This invention is based on the use of libraries of genomic probes in insitu hybridization to interphase nuclei or metaphase spreads ofchromosomes to detect and localize chromosomal abnormalities. Theseabnormalities can be of several types, including extra or missingindividual chromosomes, extra or missing portions of a chromosome(segmental duplications or deletions), breaks, rings and chromosomalrearrangements. Chromosomal rearrangements include translocations,dicentrics, inversions, insertions, amplification and deletions.

Generally, the methods of the invention consist of two steps: 1) Thecreation of a mapped library of probes, and 2) The in situ hybridizationof those probes to a chromosome and subsequent detection ofhybridization frequency to determine relative copy number of aparticular chromosomal region.

The mapped libraries of probes consist of a set of probes which whenhybridized to a normal chromosome are distributed relatively uniformlyacross the region of interest. The region typically is one chromosome ora part of one chromosome. In certain embodiments, the library of probescan encompass the entire genome.

Each probe in the library is hybridized to the normal chromosome in ametaphase spread in situ. The physical location of the probe on thechromosome is determined by visualization of a marker, as described indetail below. Probe locations are typically expressed as the averagefractional length from the p telomere (FLpter).

Once probes which hybridize to unique regions and show a relativelyuniform distribution have been identified and mapped, they may be usedto probe chromosomes of unknown genetic composition to determine thepresence or absence of amplifications or deletions and otherabnormalities. In particular, they may be used to probe interphasenuclei which is the prevalent cell stage in most tissues that are notactively dividing. Hybridization spots may be counted by regularfluorescence microscopy to give the copy number as a function of FLpter.The copy number relative to normal cells is then indicative of variouschromosome abnormalities such as amplifications, deletions and the like.

Selection of a Chromosome

Typically, the probe libraries of the present invention are derived fromlibraries spanning an entire chromosome. Alternatively, libraries areconstructed from multiple chromosomes, or from regions spanning asegment of a chromosome. Single chromosomes may be isolated by flowsorting using methods well known to those of skill in the art. Briefly,chromosomes are isolated from cells blocked in metaphase by the additione.g., colcemid and stained with two DNA-binding fluorescent dyes. Thestained chromosomes are then passed through a cell sorter and isolatedusing bivariate analysis of the chromosomes by size and base paircomposition (see, e.g., Blennow et al., Hum. Genet. 90:371-374 (1992).

One of skill would recognize that the choice of a chromosome to map maybe influenced by prior knowledge of the association of a particularchromosome with certain disease conditions. For example, chromosome 17is known to harbor several disease-linked genes including p53, RARA,NF1, CMT and ERBB and there are reports suggesting the presence of atumor suppressor gene distal to p53 (e.g. Coles, et al. Lancet 336:761-763.(1990), Cropp, et al. Proc. Natl. Acad. Sci. USA87:7737-7741.(1990) and Matsumura, et al. Cancer Res. 52: 3474-3477(1992)), a gene associated with early onset breast cancer at 17q21(Easton, et al. Am. J. Human Genet., 52:678-701 (1993)) andamplification of one or more regions in breast cancer. Kallioniemi, etal. Proc. Natl. Acad. Sci. USA 89:5321-5325 (1992).

Alternatively, whole genome screening techniques such as Southernblotting, and Comparative Genome Hybridization (CGH) may be used toidentify chromosomes subject to frequent deletion and amplificationevents and thus good candidates for further study using the presentinvention. In particular CGH provides an effective means for screeningthe genome for frequent deletion or amplification events. CGH studieshave indicated that sequences on chromosome 20q are frequently amplifiedin both breast tumor cell lines and primary breast tumors. Abnormalitiescan also be identified that are suitable for prenatal screening.

In CGH, differently labeled test DNA and normal reference DNA arehybridized simultaneously to normal chromosome metaphase spreads. Thehybridization is detected with two different fluorochromes. Abnormalchromosomal regions containing duplications, deletions or amplificationsare detected as changes in the ratio of the two fluorochromes along thetarget chromosomes. For a detailed description of CGH see Kallioniemi,et al. Science, 258: 818-821 (1992).

One of skill would recognize that a library of the present inventioncould be used to screen the entire genome. However because of the highresolution of the technique and the large number of probes required toscreen the entire genome, CGH or other methods are preferred for aninitial screening.

Production of a Probe Library

In a preferred embodiment, a selected chromosome is isolated by flowcytometry, as described above. The chromosome is then digested withrestriction enzymes appropriate to give DNA sequences of at least about20 kb and more preferably about 40 kb. Techniques of partial sequencedigestion are well known in the art. See, for example Perbal, APractical Guide to Molecular Cloning 2nd Ed., Wiley New York (1988)incorporated herein by reference. The resulting sequences are ligatedwith a vector which contains a resistance marker. The vector istransfected into and propagated in the appropriate host. Exemplaryvectors suitable for this purpose include cosmids, yeast artificialchromosomes (YACs), bacterial artificial chromosomes (BACs) and P1phage. Typically, cosmid libraries are prepared. The cosmid library thenconsists of single clones of the transfected bacteria.

While it is possible to generate cosmid libraries, as described above,libraries spanning entire chromosomes are available commercially(Clonetech, South San Francisco, Calif.) or from the Los Alamos NationalLaboratory. For example, the Los Alamos supplies a library designatedLA17NC01 which comprises a set of inserts in cosmids that span theentire chromosome 17 sorted from the mouse-human hybrid cell line,38L-27. The Los Alamos library for chromosome 20 is designated LA20NC01.

The cosmid probes must be labeled for use in in situ hybridization. Theprobes may be detectably labeled prior to the hybridization reaction.Alternatively, a detectable label may be selected which binds to thehybridization product. Probes may be labeled with any detectable groupfor use in practicing the invention. Such detectable group can be anymaterial having a detectable physical or chemical property. Suchdetectable labels have been well-developed in the field of immunoassaysand in general most any label useful in such methods can be applied tothe present invention. Thus a label is any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. Useful labels in the present invention include fluorescent dyes,electron-dense reagents, enzymes (as commonly used in an ELISA), biotin,dioxigenin, or haptens and proteins for which antisera or monoclonalantibodies are available. The particular label used is note critical tothe present invention, so long as it does not interfere with the in situhybridization of the probe. In addition the label must be detectible inas low copy number as possible thereby maximizing the sensitivity of theassay and yet be detectible above any background signal. Finally, alabel must be chosen that provides a highly localized signal therebyproviding a high degree of spatial resolution when physically mappingthe probe against the chromosome. In a preferred embodiment, the labelis digoxigenin-11-dUTP or biotin-14-dATP, which are then detected usingfluorophores.

The labels may be coupled to the probes in a variety of means known tothose of skill in the art. In a preferred embodiment the probe will belabeled using nick translation or random primer extension (Rigby, et al.J. Mol. Biol., 113:237 (1977) or Sambrook, et al., Molecular Cloning--ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1985)).

Mapping of Probe Library

Once a probe library is constructed, a subset of the probes isphysically mapped on the selected chromosome. FISH and digital imageanalysis can be used to localize cosmids along the desired chromosome.This method is described in detail below and in Lichter et al., Science,247:64-69 (1990). Briefly, the clones are mapped by FISH to metaphasespreads from normal cells using e.g., FITC as the fluorophore. Thechromosomes are counterstained by a stain which stains DNA irrespectiveof base composition (e.g., propidium iodide), to define the outlining ofthe chromosome. The stained metaphases are imaged in a fluorescencemicroscope with a polychromatic beam-splitter to avoid color-dependentimage shifts. The different color images are acquired with a CCD cameraand the digitized images are stored in a computer. A computer program isthen used to calculate the chromosome axis, project the two (for singlecopy sequences) FITC signals perpendicularly onto this axis, andcalculate the average fractional length from a defined position,typically the p-telomere.

The accuracy of the mapped positions of the probes can be increasedusing interphase mapping. Briefly, the distance between two probes whichare found by metaphase mapping to be very close in measured in normalinterphase nuclei. The genomic distance between the two is equal to thesquare of the physical distance (Van den Engh et al., Science 257:1410(1992)). If the order is uncertain, the probes are labeled withdifferent colors and their relative distance to a third (distant) probe.Trask et al., Am. J. Hum. Genet. 48:1 (1991).

Typically, a mapped library will consist of between about 20 and about125 clones, more usually between about 30 and about 50 clones. Ideally,the clones are distributed relatively uniformly across the region ofinterest, usually a whole chromosome.

In situ Hybridization with Mapped Library

The mapped library is then used to screen for chromosomal abnormalitiesin a sample. In the methods of the invention, a chromosome sample(typically either a metaphase spread or interphase nuclei) is analyzedusing standard in situ hybridization techniques. Several guides to thetechniques are available, e.g., Gall et al. Meth. Enzymol., 21:470-480(1981) and Angerer et al. in Genetic Engineering: Principles and MethodsSetlow and Hollaender, Eds. Vol 7, pgs 43-65 (plenum Press, New York1985).

Briefly, a chromosomal sample is prepared by depositing cells, either assingle cell suspensions or as tissue preparation, on solid supports suchas glass slides and fixed by choosing a fixative which provides the bestspatial resolution of the cells and the optimal hybridizationefficiency.

Generally, in situ hybridization comprises the following major steps:(1) fixation of tissue or biological structure to analyzed; (2)prehybridization treatment of the biological structure to increaseaccessibility of target DNA, and to reduce nonspecific binding; (3)hybridization of the mixture of nucleic acids to the nucleic acid in thebiological structure or tissue; (4) posthybridization washes to removenucleic acid fragments not bound in the hybridization and (5) detectionof the hybridized nucleic acid fragments. The reagent used in each ofthese steps and their conditions for use vary depending on theparticular application.

In some applications it is necessary to block the hybridization capacityof repetitive sequences. In this case, human genomic DNA is used as anagent to block such hybridization. The preferred size range is fromabout 200 bp to about 1000 bases, more preferably between about 400 toabout 800 bp for double stranded, nick translated nucleic acids.

Hybridization protocols for the particular applications disclosed hereare described in detail below. Suitable protocols are described inPinkel et al. Proc. Natl. Acad. Sci. USA, 85:9138-9142 (1988) and in EPOPub. No. 430,402.

Standard in situ hybridization techniques are used to probe a givensample. Hybridization protocols for the particular applicationsdisclosed here are described in detail below. Suitable protocols aredescribed in Pinkel et al. Proc. Natl. Acad. Sci. USA, 85:9138-9142(1988) and in EPO Pub. No. 430,402.

Typically, it is desirable to use dual color FISH, in which two probesare utilized, each labelled by a different fluorescent dye. A test probethat hybridizes to the region of interest is labelled with one dye, anda control probe that hybridizes to a different region is labelled with asecond dye. A nucleic acid that hybridizes to a stable portion of thechromosome of interest, such as the centromere region, is often mostuseful as the control probe. In this way, differences between efficiencyof hybridization from sample to sample can be accounted for.

The FISH methods for detecting chromosomal abnormalities can beperformed on nanogram quantities of the subject nucleic acids. Paraffinembedded tumor sections can be used, as can fresh or frozen material.Because FISH can be applied to the limited material, touch preparationsprepared from uncultured primary tumors can also be used (see, e.g.,Kallioniemi, A. et al., Cytogenet. Cell Genet. 60: 190-193 (1992)). Forinstance, small biopsy tissue samples from tumors can be used for touchpreparations (see, e.g., Kallioniemi, A. et al., Cytogenet. Cell Genet.60: 190-193 (1992)). Small numbers of cells obtained from aspirationbiopsy or cells in bodily fluids (e.g., blood, urine, sputum and thelike) can also be analyzed. For prenatal diagnosis, appropriate sampleswill include amniotic fluid and the like.

Once a region of interest has been identified and mapped with themethods of the invention, one of skill will recognize that there arenumerous means of identifying and/or screening for this region. Theregion may be sequenced by digesting chromosomal DNA with restrictionenzymes and identifying the specific duplication-bearing fragments usingthe mapped cosmids of the invention as hybridization probes. Thepositive clones may then be subcloned into appropriate vectors andsequenced.

Sequence information permits the design of highly specific hybridizationprobes or amplification primers suitable for detection of the targetsequences. This is useful for diagnostic screening systems as well asresearch purposes.

Means for detecting specific DNA sequences are well known to those ofskill in the art. For instance, oligonucleotide probes chosen to becomplementary to a select subsequence with the region can be used.Alternatively, sequences or subsequences may be amplified by a varietyof DNA amplification techniques (for example via polymerase chainreaction, ligase chain reaction, transcription amplification, etc.)prior to detection using a probe. Amplification of DNA increasessensitivity of the assay by providing more copies of possible targetsubsequences. In addition, by using labeled primers in the amplificationprocess, the DNA sequences may be labeled as they are amplified.

The following example is provided to illustrate but not limit thepresent invention.

EXAMPLE 1 Mapping of Chromosome 20

Results of experiments employing "Comparative Genome Hybridization"(CGH) to screen the whole genome for amplifications, indicate thatsequences on chromosome 20q are frequently amplified in both breasttumor cell lines and primary breast tumors. In order to define thesegenetic alterations in more detail, a library of cosmid FISH probes wasisolated and physically mapped to chromosome 20. The library of mappedprobes could then be used to probe chromosome 20 using FISH to determinethe particular loci involved in amplifications and deletions. Thisexample details the creation of the library of probes physically mappedto chromosome 20.

Cosmids from a chromosome 20 library were isolated at random from singlebacterial clones using Qiagen columns according to the manufacturersinstructions (Qiagen Inc., Chatsworth, Calif.). Cosmid DNA was labeledby nick-translation with biotin-14-dATP (Gibco), to give fragments oflength 0.3-1.0 kb (under non-denaturing conditions). These probes werehybridized to normal human lymphocyte metaphase preparations. The slideswere denatured at 70° C. for 3 minutes in 70% formamide/2X SSC, followedby dehydration in 70%/85%/100% ethanol. The slides were hybridizedovernight with 40 ng of human biotin-labeled cosmid DNA in the presenceof 5 μg human placental DNA (Sigma) in 10 μl 50% formamide/2X SSC at 37°C. The probe was denatured at 70° C. for 5 minutes and allowed torenature at 37° C. prior to application to the slides. The slides werewashed three times in 50% formamide/2X SSC, once in 0.1X SSC, and twicein 2X SSC at 45° C. (15 minutes for each wash). The remaining steps wereperformed at room temperature.

The slides were equilibrated in 4X SSC/0.1% Triton X100 ("wash" buffer),and blocked for 5 minutes in wash buffer with 5% dry milk/0.1% SodiumAzide ("block" buffer). Staining for biotinylated probe was done with 5μg/ml avidin-FITC (60 minutes, in block buffer), amplified by 30 minutesincubation with 5 μg/ml biotinylated anti-avidin (in block buffer), andanother 30 minutes incubation with avidin-FITC. The slides were thenwashed three times for 10 minutes each after each staining step. Theslides were equilibrated in 0.1X SSC prior to application of anti-fadesolution (ref) with 0.05 μg/ml propidium iodide and 0.4 μM DAPI.

The slides were first inspected in a fluorescence microscope todetermine whether signals were present (29/40 cosmids tested) and, ifso, whether the cosmid detected single-copy sequences (28/40 cosmidstested). One cosmid hybridized to the centromere of chromosome 20, butalso hybridized to the p-arms of acrocentric chromosomes. Metaphaseshybridized with cosmids detecting single copy sequences were analyzed ina Nikon SA fluorescence microscope equipped with a CCD camera(Photometrics Inc., Tucson, Ariz.) and a polychromatic beamsplitter(Chroma Technology Inc., Brattleborrough, Vt.) to avoid color-dependentimage shifts. The images of chromosome 20 were analyzed with computersoftware to determine the mean position (of the two FITC probe spots) interm of fractional length from the p telomere (FLpter) along thechromosome axis as defined by propidium iodide staining using themethods generally described by Lichter et al., supra.

Several metaphases were analyzed for each cosmid, the FLpter value andthe SEM are given in Table 1. The average SD of all the FLpter valueswas 0.030, corresponding to -2 Mb. Cosmids which are separated by aFLpter value of less than 2.5 times the SEM could not be ordered withstatistical confidence. The vertical lines in Table 1 span cosmids whichcould not be ordered reliably.

                  TABLE 1                                                         ______________________________________                                        FLpter values of chromosome 20 cosmids.                                       Cosmid       FLpter     n       SEM                                           ______________________________________                                         ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                     ______________________________________                                         *Any two cosmids connected by a vertical line cannot be ordered with          statistical confidence.                                                  

The FLpter values are graphically illustrated in FIG. 1, together withan ideogram of chromosome 20. A chromosome 20 centromere probe (p3-4)mapped to FLpter=0.443 (SEM=0.008, n=10), in good agreement with theposition of the centromere in this ideogram and the reported physicallocation of the chromosome 20 centromere (Schnittger, et al. Genomics,16:50-55 (1993); Passarge, E. pp. 135-205 In Methods in Human GeneticsSchwarzacher & Wolf, Eds. Springer, Berlin (1974). The correspondingband locations indicated in FIG. 1 were also confirmed visually relativeto the position of DAPI bright bands (corresponding approximately toGiemsa-stained bands under these conditions). The cosmids seem to befairly evenly distributed over the whole chromosome, with possibleunder- and over-representation in the centromere region and the regionwith a FLpter value of -0.6, respectively.

Mapping Chromosome 17

The chromosome 17 cosmid library (designated LA17NC01) was prepared atthe Los Alamos National Laboratory from chromosomes flow sorted from themouse-human hybrid cell line, 38L-27. The sorted chromosomes wereexamined for purity using FISH. DNA was extracted, partially digestedwith Sau3A1, dephosphorylated, and cloned into sCosl with HB101 as thehost. Characterization of the initial library showed it to have 39Xrepresentation with 92% human inserts, 2.6% mouse and 5.4%non-recombinant.

Approximately 12,000 colonies were picked and grown in microtiter platesto provide a 5X coverage of chromosome 17. The contents of one set ofthese plates were pooled together to provide a pooled 5X library. Forthis study, the 5X library was plated and 288 individual clones werepicked at random and arrayed in three microtiter plates. Seventy one ofthese were analyzed using FISH. Twenty cosmid probes previously mappedby genetic linkage analysis (O'Connell, et al. Genomics, 15:38-47 (1993)also were selected for analysis.

FISH was performed essentially as described previously. See Kallioniemi,et al. Proc. Natl. Acad. Sci. USA, 89:5321-5325 (1992) and Pinkel, etal., Proc. Natl. Acad. Sci. USA, 85:9138-9142 (1988) both of which areincorporated herein by reference. Briefly, DNA was isolated fromindividual clones and from the 5X pool using Qiagen columns (Qiagen Inc.Chatsworth, Calif.) according to the manufacturers instructions. DNA waslabeled using nick translation with biotin-14-dATP. DNA from achromosome 17 centromeric repeat probe was labeled withdigoxigenin-11-dUTP. Each probe was hybridized along with the chromosome17 centromere probe to metaphase spreads prepared from normal peripheralblood lymphocytes. Hybridized probes were detected using Avidin-TexasRed and anti-digoxigenin-FITC. Metaphase chromosomes were counterstainedusing DAPI in an anti-fade solution.

Analysis of the samples was accomplished using a digital image analysissystem as described above. A semi-automated program was used to 1)segment each DAPI image, 2) define the chromosome medial axis, 3) definethe center of mass of each candidate hybridization domain, and 4)calculate the fractional location of each domain along the chromosomeaxis relative to the telomere of the short arm of the chromosome(FLpter). Candidate hybridization domains defined by the analysisprogram were confirmed by visual inspection. A chromosome 17 centromericprobe was used in each hybridization as an internal reference. If theFLpter value of the centromere did not fall within the expected range(0.300<FLpter<0.342), the chromosome was not used for FLptercalculation.

FISH using DNA from the whole library (LA17NC01) as a painting proberesulted in intense, specific staining of the entire chromosome 17indicating that all portions of the chromosome are represented in thelibrary. Seventy-one individual cosmids selected from the library wereroughly mapped using FISH. Of these, 15 (21%) mapped to the p-arm, 46(65%) to the q-arm, 2 (3%) to the peri-centromeric repeat region. Thisdistribution is approximately that expected for randomly distributedprobes. Eight cosmids (11%) gave no signal on any human chromosome andso may have been of non-human origin. This is consistent with theinitial library characterization by slot blot analysis that showed 8% ofthe clones to be non recombinant or to contain a mouse insert.

In order to identify probes that would be useful for FISH analysis ofinterphase cells, the 40 cosmids that gave the most distinctivehybridization signals both in metaphase and interphase were mapped usingdigital image analysis. FLpter values are listed in Table 2 for eachcosmid. The standard deviation (sd) for the FLpter measurements and thenumber of chromosomes analyzed for each probe (n) are listed to permitcalculation of the standard error of the mean (sem=sd√/n) for eachFLpter estimate. Probes whose FLpter means differ by >2.5 sem can beordered with statistical confidence.

FIG. 3 shows that the probes are distributed over the whole chromosome,although there seems to be a slight over-representation of sequencesnear 17p11-p12. Two color, pair-wise hybridizations to metaphasechromosomes showed that these did not represent the same clone. FLptervalues are related to the ICSN chromosome 17 ideogram in FIG. 3 sinceother studies have shown a reasonable correspondence between FLptervalue and band location (Lawrence, et al., Science, 249:928-932 (1990);Lichter, P., et al. Science 247:64-69. (1990)). However, this relationshould be considered only approximate since band locations on theideograms are inexact.

Twenty cosmids that had also been mapped by genetic linkage analysis(O'Connell, et al. Genomics, 15:38-47 (1993)) were also physicallymapped. The average standard deviation of the FLpter measurements forall cosmids was 0.035. This corresponds to about 3 Mbs. The averagemapping precision determined as the standard error of the mean (sem) wasabout 0.01 corresponding to ˜1 Mb. Cosmids separated by ˜2.5 Mb (i.e.whose means are separated by >2.5 sem in Table 2) can be ordered withstatistical confidence.

Detection of Amplifications in Chromosome 20

FIG. 2, shows an example where the spot numbers of the different mapped,chromosome 20-specific cosmids (see FIG. 1) were counted in interphaseBT474 breast cancer cells using standard FISH techniques as describedabove. A region around FLpter-0.85 is heavily amplified.

All of the references cited herein are hereby incorporated by reference.For the purposes of clarity and understanding, the invention has beendescribed in these examples and the above disclosure in some detail. Itwill be apparent, however, that certain changes and modifications may bepracticed within the scope of the appended claims.

                                      TABLE 2                                     __________________________________________________________________________    FLpter values measured for 60 cosmids including 20 that were mapped           previously by genetic linkage analysis (shown in bold type).                                 Gen                Gen                                         Probe                                                                              FLpter                                                                            sd n  loc (cM)                                                                           Probe                                                                              FLpter                                                                            sd n loc (cM)                                    __________________________________________________________________________    cK17.79                                                                            0.025                                                                             0.018                                                                             5      cK17.16                                                                            0.564                                                                             0.048                                                                             8                                            cLS17.6                                                                            0.066                                                                             0.037                                                                            25 13   fLB17.20                                                                           0.580                                                                             0.046                                                                            21                                                                              90                                          fLB17.8                                                                            0.068                                                                             0.037                                                                            16 15   cK17.11                                                                            0.591                                                                             0.035                                                                            12                                            cK17.29                                                                            0.068                                                                             0.031                                                                            17      cLS17.13                                                                           0.594                                                                             0.040                                                                            22                                                                              90                                          fLB17.9                                                                            0.073                                                                             0.037                                                                            20 19   cK17.87                                                                            0.598                                                                             0.026                                                                             8                                            fLB17.16                                                                           0.076                                                                             0.041                                                                            28  15-18*                                                                            cK17.30                                                                            0.618                                                                             0.041                                                                             9                                            cK17.22                                                                            0.122                                                                             0.020                                                                            11      cK17.76                                                                            0.657                                                                             0.019                                                                            12                                            cK17.88                                                                            0.177                                                                             0.042                                                                             9      cK17.84                                                                            0.666                                                                             0.024                                                                             9                                            cK17.31                                                                            0.197                                                                             0.036                                                                            10      cK17.73                                                                            0.714                                                                             0.037                                                                            11                                            cK17.24                                                                            0.199                                                                             0.028                                                                            11      cK17.33                                                                            0.728                                                                             0.039                                                                            13                                            cK17.80                                                                            0.205                                                                             0.035                                                                            11      fLB17.4                                                                            0.773                                                                             0.033                                                                            20                                                                              111-123*                                    cK17.17                                                                            0.219                                                                             0.044                                                                             8      cK17.72                                                                            0.814                                                                             0.037                                                                            11                                            cK17.83                                                                            0.220                                                                             0.043                                                                            10      cK17.14                                                                            0.823                                                                             0.026                                                                            11                                            cK17.19                                                                            0.224                                                                             0.031                                                                            10      fLB17.14                                                                           0.825                                                                             0.033                                                                            25                                                                              120                                         pYNM67                                                                             0.233                                                                             0.057                                                                            15  59-63*                                                                            cK17.28                                                                            0.837                                                                             0.052                                                                            16                                            cK17.81                                                                            0.236                                                                             0.037                                                                            11      cK17.53                                                                            0.867                                                                             0.039                                                                            15                                            fLB17.5                                                                            0.251                                                                             0.040                                                                            17 61-64                                                                              cK17.12                                                                            0.871                                                                             0.023                                                                            12                                            cK17.23                                                                            0.306                                                                             0.029                                                                             4      cK17.54                                                                            0.894                                                                             0.032                                                                            16                                            cK17.75                                                                            0.355                                                                             0.038                                                                             9      cK17.15                                                                            0.919                                                                             0.035                                                                            12                                            fLB17.18                                                                           0.410                                                                             0.035                                                                            20 77   c1-26                                                                              0.922                                                                             0.035                                                                            18                                                                              144                                         cK17.37                                                                            0.411                                                                             0.033                                                                             5      fLB17.17                                                                           0.942                                                                             0.037                                                                            25                                                                              148-151*                                    cK17.18                                                                            0.433                                                                             0.044                                                                            12      cK17.27                                                                            0.943                                                                             0.032                                                                            13                                            cK17.32                                                                            0.446                                                                             0.030                                                                             9      cEFD52                                                                             0.950                                                                             0.045                                                                            15                                                                              167                                         fLB17.6                                                                            0.451                                                                             0.040                                                                            18  77-80*                                                                            cK17.71                                                                            0.953                                                                             0.028                                                                            11                                            cK17.89                                                                            0.470                                                                             0.048                                                                            12      cLS17.9                                                                            0.962                                                                             0.027                                                                            21                                                                              159                                         cK17.86                                                                            0.510                                                                             0.035                                                                             8      fLB17.7                                                                            0.964                                                                             0.026                                                                            24                                                                              165-169*                                    cK17.25                                                                            0.515                                                                             0.023                                                                             8      fLB17.2                                                                            0.969                                                                             0.030                                                                            28                                                                              166-170*                                    cK17.38                                                                            0.537                                                                             0.031                                                                            14      fLB17.10                                                                           0.985                                                                             0.016                                                                            23                                                                              177                                         cK17.74                                                                            0.539                                                                             0.036                                                                             9      cK17.77                                                                            0.989                                                                             0.015                                                                             9                                            fLB17.1                                                                            0.557                                                                             0.038                                                                            19 88   cK17.78                                                                            0.994                                                                             0.005                                                                            12                                            __________________________________________________________________________     *Genetic locations for these probes were estimated graphically from           information presented by 'Connell, et al. Genomics, 15: 38-47 (1993).    

What is claimed is:
 1. A method of detecting a chromosome abnormality ina preselected chromosome, the method comprising:providing a mappedlibrary of labeled probes specific to the chromosome; contacting achromosome sample from a patient with the library under conditions inwhich the probes bind selectively with target polynucleotide sequencesin the sample to form hybridization complexes; detecting thehybridization complexes; and determining the copy number of eachcomplex, thereby detecting the presence or absence of a chromsomeabnormality in the chromosome.
 2. The method of claim 1, wherein thechromosome abnormality is a deletion.
 3. The method of claim 1, whereinthe chromosome abnormality is an amplification.
 4. The method of claim1, wherein the probes are labeled with digoxigenin or biotin.
 5. Themethod of claim 1, wherein the step of detecting the hybridizationcomplexes is carried out by detecting a fluorescent label.
 6. The methodof claim 5, wherein the fluorescent label is FITC.
 7. The method ofclaim 1, wherein the hybridization complexes are detected in interphasenuclei in the sample.
 8. The method of claim 1, further comprisingcontacting the sample with a reference probe which binds selectively toa sequence within the centromere of the preselected chromosome.
 9. Amethod of detecting an amplification at about position FLpter 0.85 onhuman chromosome 20, the method comprising:contacting a chromosomesample from a patient with a composition consisting essentially of oneor more labeled nucleic acid probes each of which binds selectively to atarget polynucleotide sequence at about position FLpter 0.85 on humanchromosome 20 under conditions in which the probe forms a stablehybridization complex with the target sequence; detecting thehybridization complex, thereby detecting the presence or absence of theamplification.
 10. The method of claim 8, wherein the step of detectingthe hybridization complex comprises determining the copy number of thetarget sequence.
 11. The method of claim 8, wherein the probe is labeledwith digoxigenin or biotin.
 12. The method of claim 8, wherein the probeis selected from the group consisting of polynucleotide sequences fromcS20.10A1, cS20.10B5, cS20.10H1, and cS20.10E2.
 13. The method of claim8, wherein the hybridization complex is detected in interphase nuclei inthe sample.
 14. The method of claim 8, further comprising contacting thesample with a reference probe which binds selectively to chromosome 20centromere.
 15. A composition comprising a nucleic acid probe whichbinds selectively to a target polynucleotide sequence at about FLpter0.85 on human chromosome
 20. 16. The composition of claim 14, whereinthe probe is labelled with digoxigenin or biotin.
 17. The composition ofclaim 14, wherein the probe is selected from the group consisting ofpolynucleotide sequences from cS20.10A1, cS20.10B5, cS20.10H1, andcS20.10E2.